RNAi mechanism plays a major role in silencing the expression of target genes by siRNAs. In the current study, in silico properties of 30 genes in omega-2 gliadin and 266 nt and 326 nt mutations were investigated before and after cloning in an expression vector. Specific primers were designed for 30 genes with spacer regions of 75 nt and 178 nt (for gene invert repeats). The frequency of siRNA site and nucleotide mutations A/U to G/C were identified for target mRNA using in silico analysis. The results showed that invert repeat sequences consisting of spacer region of 178 nt had a high efficiency for target gene without nucleotide mismatches from A/U to G/C. Spacer regions were designed with long fragments for RNAi cassette instead of intron sequences. Paying enough attention to observe the location of mismatch nucleotides in siRNA and length spacer region before and after cloning reduced the deletion time of intron. At the same time, removal of epitopes in wheat dependent exercise-induced anaphylaxis (WDEIA) and celiac disease could be considered as advantages of the applied method compared to ligation in the bacterial vector.